The consumption of foods rich in antioxidant compounds is well known to reduce the incidence of many chronic disease states, such as cancer. Moreover, the intake of certain antioxidants, such as vitamins C (ascorbic acid) and E (xcex1-tocopherol), is essential for survival in humans and most mammals, because these compounds are not biosynthesized.
Aerobic organisms are partially protected against oxidative challenges by sophisticated antioxidant defense systems. The importance of antioxidant defense systems in humans is demonstrated by the essential in vivo presence of both enzymatic and non-enzymatic antioxidant components. Oxidative stress and resultant oxidative damage may occur as a result of oxidative challenges such as air pollution or the xe2x80x9coxidative burstxe2x80x9d associated with activated neutrophils mediated by the immune response. A constant source of oxidative stress results from formation of superoxide anions via xe2x80x9celectron leakagexe2x80x9d in the mitochondria during production of adenosine triphosphate (ATP). Although a superoxide anion is not exceedingly reactive in and of itself, it can initiate a cascade of events that eventually results in the formation of highly reactive free radicals and other oxidants. If these reactive oxygen species are not controlled by enzymatic and/or non-enzymatic antioxidant systems, in vivo oxidation of critical cellular components such as membranes, DNA, and proteins will result, eventually leading to tissue damage and dysfunction.
Inflammation can be induced by acute exercise in untrained individuals (Jenkins, R. R. et al. [1993] Med Sci Sports Exerc 25:213-7). Strenuous exercise increases oxygen consumption and causes disturbance of the intracellular homeostasis between pro-oxidants and antioxidant, resulting in oxidative stress. Reactive oxygen species pose a serious threat to the cellular antioxidant defense system, such as by diminishing the reserve of antioxidant vitamins and glutathione, and increasing tissue susceptibility to oxidative damage. However, enzymatic and non-enzymatic antioxidants have demonstrated great adaptation to acute and chronic exercise. The delicate balance between pro-oxidants and antioxidants suggests that supplementation of antioxidants may be desirable for physically active individuals under certain physiological conditions by providing a larger protective margin.
In vitro experiments have shown that certain substances, including some antioxidant vitamins, such as vitamin C, are reactive with free iron and can cause oxidative damage to biomolecules. Under normal physiological conditions, metals are found bound to circulating proteins and rendered redox-inactive (Halliwell, B. et al. [1999] Free radicals in Biology and Medicine. Oxford N.Y.: Clarendon Press, Oxford University Press; Winterboum, C. C. [1981] Biochem J 198:125-31). However , levels of free metals, such as iron, may be elevated during acute inflammation and become redox-active. Others have shown that under several physiological and pathophysiological conditions, increases in free iron in the presence of vitamin C results in oxidative stress.
Inflammation stimulates polymorphonuclear leukocytes and macrophages that produce large amounts of superoxide (O2xe2x80xa2xe2x88x92) and hydrogen peroxide (H2O2) (Babior, B. M. et al. [1973] J Clin Invest 52:741-744; Halliwell, B., et al. [1999] supra). The detrimental effects of these radicals may be amplified in the presence of iron and the subsequent formation of other reactive intermediates, such as the hydroxyl radical (HOxe2x80xa2). NADPH oxidase, a membrane-associated electron transport chain protein, becomes activated during inflammation and directly reduces O2 to O2xe2x80xa2xe2x88x92 (Equation 1). Superoxide can then be dismutated by superoxide dismutase to produce H2O2 (Equation 2).
NADPH+2O2xe2x86x922O2xe2x80xa2xe2x88x92+NADP++H+xe2x80x83xe2x80x83(Equation 1)
O2xe2x80xa2xe2x88x92+O2xe2x80xa2xe2x88x92+2H+xe2x86x92H2O2+O2xe2x80x83xe2x80x83(Equation 2)
Superoxide can reduce transition metals, including ferric iron (Fe3+), to ferrous iron (Fe2+) (Equation 3). The reduced metal ion can then react with H2O2 to generate the highly oxidizing HOxe2x80xa2 radical species (Equation 4).
Fe3 ++O2xe2x80xa2xe2x88x92xe2x86x92O2+Fe2+xe2x80x83xe2x80x83(Equation 3)
Fe2++H2O2xe2x86x92HOxe2x80xa2+OHxe2x88x92+Fe3+xe2x80x83xe2x80x83(Equation 4)
The hydroxyl radical has been widely postulated to cause significant damage to several biomolecules in vivo. Although the relevance of the hydroxyl radical in biology has been questioned because of the requirement of redox-active free iron, Biemond and colleagues have shown iron release from ferritin during inflammation (Biemond, P. et al. [1984] J Clin Invest 73(6): 1576-9).
In vitro vitamin C can exert pro-oxidant effects. The reduction potentials of Fe3+ (xe2x88x920.4 V) and ascorbate (xe2x88x920.17 V) easily allow the formation of the ascorbate radical and Fe2+ iron (Equation 5).
Fe3++ascorbatexe2x86x92Fe2++ascorbatexe2x80xa2xe2x80x83xe2x80x83(Equation 5)
In addition, the formation of ferrous iron increases the possibility of the production of HOxe2x80xa2 by reacting with H2O2 (Equation 4). Thus, it is feasible that the release of iron and the presence of vitamin C during acute inflammation characterized by high fluxes of oxidants could lead to HOxe2x80xa2 and ascorbate generation.
The reduction potentials of Fe3+ (xe2x88x920.4 V) and ascorbate (xe2x88x920.17 V) easily allow the formation of the ascorbate radical and Fe2+ iron (Equation 5). Therefore, in vitro vitamin C can exert pro-oxidant effects, by converting Fe3+ into Fe2+, which reacts with H2O2 to generate HOxe2x80xa2 (Halliwell, B. et al. [1999] supra; Roginsky, V. A. et al. [1994] Free Radic Biol Med 17:93-103). Iron-ascorbate mixtures have been shown to stimulate free-radical damage to DNA, lipids, and proteins in vitro (Halliwell, B. et al. [1990] Methods Enzymol 186:1-85). In vivo iron (Kadiiska, M. B. et al. [1995] J Clin Invest 96:1653-7) supplementation and ascorbate-copper supplementation (Kadiiska, M. B. et al. [1992] Mol Pharmacol 42:723-9) to rats have been reported to stimulate HOxe2x80xa2 generation. Thus, it is feasible that the release of iron and the presence of vitamin C during acute inflammation could lead to HOxe2x80xa2 and ascorbatexe2x80xa2 generation.
Until recently, there has been no investigation as to whether antioxidant supplements can act as pro-oxidants, causing oxidative stress in humans or animals under acute inflammatory conditions characterized by increases in levels of redox-active metal ions. Long-term supplementation with these compounds could have detrimental effects in patients suffering from acute inflammation and inflammatory conditions accompanied by increased levels of free iron. However, these supplements could also have detrimental effects on patients with certain disease conditions characterized by increased levels of free iron without the presence of inflammation. Such disease conditions include, for example, Alzheimer""s, Parkinson""s, atherosclerosis, diabetes, and hemachromotosis (Gerlach, M. et al. [1994] J Neurochem 63(3):793-807; Halliwell, B. et al. [1985] Mol Aspects Med 8(2):89-193). One study protective effect of vitamin C in the plasma in the presence of high levels of free iron in vitro, without the presence of inflammation (Berger, T. M. et al. [1997] J Biol Chem 272: 15656-60). The Berger study (Berger, T. M. et al. [1997] supra) investigated if the naturally occurring high levels of ascorbic acid in pre-term infants, along with the presence of detectable levels of iron, would increase oxidative stress. The Berger study showed no concurrent increases in either lipid hydroperoxides or protein carbonyls in the plasma of these infants. In contrast, others suggest that co-supplementing healthy volunteers with iron and vitamin C increased levels of oxidative DNA damage in white blood cells. It was concluded that increased levels of DNA damage in well-nourished subjects after iron/ascorbate supplementation are disturbing in view of the frequent use of dietary supplements containing both iron salts and ascorbate (Bolann, B. J. et al. [1990] Eur J Biochem 193:899-904; Rehman et al [1998] Biochem Biophys Res Commun 246:293-8).
Inflammation can be induced by prolonged or damaging exercise and can increase the levels of free iron (Jenkins, R. R. et al. [1993] supra). Part of the inflammatory reaction to muscle injury includes a systemic response in addition to the changes observed locally at the muscle. Several types of exercises damage enzymes and lipid membranes, increase DNA damage, stimulate oxidative stress, and increase plasma markers of cell damage (Jenkins, R. R. et al. [1993] supra; Camus, G. et al. [1994] Arch Int Physiol Biochim Biophys 102:67-70; Fielding, R. A. et al. [2000] Med Sci Sports Exerc 32:359-64; Ji, L. L. [1995] Exerc Sport Sci Rev 23:135-66; Leeuwenburgh, C. et al. [1999] Free Radic Biol Med 27:186-92; MacIntyre, D. L. et al. [2000] Eur J Appl Physiol 81:47-53; Maughan, R. J. et al. [1989] Muscle Nerve 12:332-6; Powers, S. K. et al. [1999] Med Sci Sports Exerc 31:987-97).
Eccentric exercise is a particularly damaging activity involving forced lengthening of a muscle as it develops tension and leads to a condition characterized by severe inflammation and edema (MacIntyre, D. L. et al. [2000] supra). Most exercise encountered is concentric/eccentric exercise. Purely eccentrically based exercise is rarely encountered in sports or in activities of daily living. Examples of eccentric exercises are downhill running and eccentric arm exercises, which have been shown to increase neutrophil migration into the skeletal muscle after such injury (Belcastro, A. N. et al. [1996] J Appl Physiol 80:1331-5; Camus, G. et al. [1994] supra; Fielding, R. A. et al. [2000] supra; MacIntyre, D. L. et al. [2000] supra). In addition, it appears that eccentric exercise may produce a cellular environment of acute phase inflammation characterized by increases in levels of redox-active metal ions.
The influence eccentric exercise has on some biological markers of inflammation, oxidative stress, and muscle injury has been studied. These markers include, for example, myeloperoxidase, interleuken-6 (IL-6) , lactate dehydrogenase (LDH), creatine kinase (CK), myoglobin, and total antioxidant capacity. Some of these investigations examined the effect of particular antioxidant supplements (e.g., vitamins C and E) and specific non-steroidal anti-inflammatory drugs (NSAIDS) on some of these markers before and/or after various degrees of eccentric exercise (Van der Meulen et al. [1997] J Applied Physiology 83(3):817-23; Maxwell et al. [ 1993] Free Radic Res Commun 19(3):191-202; Pizza et al. [1999] Int J Sports Med 20:-102; Bourgeois et al. [1999] Med and Science in Sports and Exercise 31(1):4-9; and Croisier et al. [1996] Mediators of Inflammation 5(3):230-4).
Currently, no reliable human or animal model exists to rapidly test substances, such as drugs and natural compounds, for anti-/pro-inflammatory or anti-/pro-oxidant properties. Most substances tested on humans are tested on a specific population suffering from an inflammatory disease condition, e.g., arthritis. Unfortunately, each individual""s symptoms are vastly different and responses are highly variable, making objective testing difficult and results unreliable. In addition, there is currently no method for testing such compounds under conditions of acute inflammation characterized by increases in redox-active metal ions. Therefore, it would be advantageous to provide a reliable method for testing a substance on healthy subjects in order to discern the substance""s inflammatory and/or oxidant properties under acute inflammatory conditions and/or under conditions of elevated redox-active metal ions.
The subject invention provides methods and systems for testing a substance for inflammatory or oxidant properties. In a preferred embodiment, the method comprises the steps of applying an eccentric exercise stimulus to a subject, thereby inducing a muscle injury in the subject; administering a substance to the subject; measuring at least one biological marker from the subject, wherein the biological marker(s) are selected from the group consisting of inflammatory markers, oxidative stress markers, cell damage markers, and combinations thereof; and correlating the measurements of the biological marker(s) with the inflammatory or oxidant properties of the substance.
The subject invention also provides a system for determining the inflammatory or oxidant properties of a substance comprising sampling means for obtaining a biological sample from a subject; stimulus means for administering eccentric exercise to the subject; analysis means for measuring the amount of at least one biological marker in the biological sample, wherein the biological marker(s) are selected from the group consisting of inflammatory markers, oxidative stress markers, and cell damage markers, or combinations thereof; and means for correlating the measurements of the biological markers with the inflammatory or oxidant properties of the substance.
Advantageously, the muscle injury resulting from the eccentric exercise stimulus induces an acute inflammatory response and causes increased levels of redox-active metal ions within the subject. The method and system of the subject invention can be used to determine if a substance has pro-inflammatory or anti-inflammatory properties, as well as pro-oxidant or antioxidant properties.
The biological marker(s) measured according to the subject invention may be, for example, free iron, total antioxidant status, 8-isoprostane (8-Iso-PGF2xcex1), superoxide dismutase (SOD), glutathione peroxidase (GPX), lactate dehydrogenase (LDH), C-reactive protein, lipid hydroperoxidase (LOOH), myeloperoxidase, interleukin-6 (IL-6), creatine kinase (CK), dityrosine, and 8-hydroxyguanine, or combinations thereof. Preferably, the biological marker(s) are selected from the group consisting of free iron, 8-Iso-PGF2xcex1, SOD, GPX, dityrosine, and 8-hydroxyguanine, or combinations thereof. More preferably, the biological marker(s) are selected from the group consisting of free iron, 8-Iso-PGF2xcex1, SOD, and GPX, or combinations thereof. In a specific embodiment, the biological markers are free iron and/or 8-Iso-PGF2xcex1.
The eccentric exercise stimulus applied to the subject may be, for example, an eccentric arm curl exercise, an eccentric leg curl exercise, and a downhill running exercise, or combinations thereof. In a specific embodiment, the eccentric exercise stimulus is applied at between about 60% and about 95% of the subject""s maximum intensity. In another embodiment, the eccentric exercise stimulus is applied at between about 70% and about 90% of the subject""s maximum intensity. In a further embodiment, the eccentric exercise stimulus is applied at about 80% of the subject""s maximum intensity. In another embodiment, the maximum intensity of the eccentric exercise stimulus is the subject""s one-repetition maximum weight equivalent (e.g., eccentric press weight, eccentric curl weight, or the magnitude of other force applied to the subject""s body), or the subject""s maximum heart rate.